The overarching aim of this proposal is to understand the design principles of the regulatory network that regulates mitosis. The main focus is on the bistable trigger that governs the transition from interphase to mitosis. The proposed approaches include quantitative experimental studies in HeLa cells; Xenopus egg extracts, and reconstituted in vitro systems; fluorescent biosensor development; and computational studies. There are three specific aims: (1) To determine whether a bistable switch triggers the translocation of active cyclin B1/Cdk1 from the cytoplasm to the nucleus. Quantitative studies of the timing of early mitotic events in HeLa cells have led us to hypothesize that cyclin B1 is driven into the nucleus by a bistable circuit, where nuclear cyclin B1/Cdk1 complexes promote the import and/or inhibit the export of more cyclin B1/Cdk1 complexes. This hypothesis will be tested through experimental studies, and the implications of this hypothesis for noise propagation and self-organization will be explored through computational studies. (2) To determine how robust bistability is achieved by the bistable Cdk1/Cdc25/Wee1 system. Recent computational studies show that the striking 'mirror image' relationship between Cdc25 and Wee1 has the potential to render the bistability of the Cdk1/Cdc25/Wee1 nearly impervious to changes in protein concentration. We plan to test this hypothesis through reconstitution of the bistability switch in vitro and through perturbation of the system in extracto. (3) To develop a new class of fluorescent protein kinase sensors by exploiting the changes in bulk electrostatic properties that can occur when a protein is phosphorylated. PUBLIC HEALTH RELEVANCE: The ability of a cell to replicate itself through growth, mitosis and cell division is one of the most basic, fundamental aspects of life. An understanding of the process also promises to shed light on cancer and cancer chemotherapy.